Protease activities are known to be critical in the pathogenesis of a wide variety of diseases including cancer, cardiovascular disease, and inflammatory and infectious diseases. Although large-scale efforts are providing valuable information regarding the expression of many diverse proteases in normal and disease tissues, these methods do not report the functional activity of proteases. Protease beacons are emerging as a powerful tool to non-invasively measure protease activity in vivo, and will benefit from approaches that enable more selective cleavage by individual targeted proteases and probe retention at the site of activation. This project aims to validate a generic molecular probe design to detect, localize, and quantify protease activities in vivo in normal and pathologic tissues and whole organisms. Specifically, a new class of protease activity probes will be created that exhibit the desired characteristics of high tissue uptake, selective activation by individual target proteases, and accumulation at sites of activation. The utility of these probes for the localization and quantification of protease activities in vivo will be demonstrated in normal mice and the ApoE(-/-) mouse model of atherosclerosis. Completion of this project will yield probes that accurately and reliably report the activity of proteases involved in normal and pathologic processes, with high potential for translational to humans. More generally, successful completion of the aims of this proposal will open the door to the measurement and localization of a wide range of protease activities in living organisms with high spatial and temporal precision.